Characterisation of a high-frequency gene encoding a strongly antigenic cystatin-like protein from Trichinella spiralis at its early invasion stage

Background: The intestinal phase is the early invasion stage of Trichinella spiralis (T. spiralis), in which muscle larvae invade intestine epithelial cells and then develop into adult worms to breed newborn larvae. Thus, intestinal infective larvae are first exposed to the immune system of the host, and antigens from the worms may be the earliest marker in the diagnosis of trichinellosis and may contribute to vaccine development to prevent Trichinella infections in pigs. Methods: A cDNA library of intestinal infective larvae of T. spiralis at 6 hours post infection (p.i.) was constructed and immunoscreened using serum collected from pigs that were infected with T. spiralis at 26 days p.i.T. spiralis cystatin-like protein (Ts-CLP) gene encoding a 45.9 kDa protein was cloned and expressed in Escherichia coli. The rabbit antisera were generated and used to determine the location of Ts-CLP in the parasite. Transcription levels of Ts-CLP in different developmental stages of T. spiralis were observed by RT-PCR. The potential application of recombinant Ts-CLP in diagnosis against T. spiralis infection was tested by ELISA. The immune protection of recombinant Ts-CLP protein against T. spiralis infection was evaluated in mice. Results: Thirty-three positive clones were selected from cDNA library, among which 20 clones encoded the same novel cystatin-like protein (Ts-CLP). Immunolocalisation and real-time quantitative PCR revealed that native Ts-CLP was localised primarily to beta-stichocytes and that the Ts-clp gene was transcribed and expressed in all developmental stages of T. spiralis. The recombinant protein rTs-CLP was recognised by pig antiserum as early as 15 days p.i., and could induce protective immunity in mice, with a 61.21% reduction in the number of muscle larvae. Conclusions: These data preliminarily suggested that Ts-CLP may play an important role in the early infection of T. spiralis and that recombinant Ts-CLP protein is a candidate antigen for diagnosis and vaccine development in Trichinella infections.